Retention index standards for liquid chromatography

ABSTRACT

A liquid chromatography method for identifying an analyte of interest utilizing as retention index standards a homologous series of neutrally charged compounds having at least one functional group bearing a positive charge and at least one functional group bearing a negative charge. The method is especially useful for liquid chromatography-mass spectrometry (LC-MS) methods, more especially for LC-MS methods employing electrospray (ESI) or atmospheric pressure chemical ionization (APCI) ionization systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/261,954 filed Sep. 12, 2014, which is a national entry ofInternational Patent Application PCT/CA2013/000252 filed Mar. 15, 2013and claims the benefit of U.S. provisional patent application U.S. Ser.No. 61/611,760 filed 16 Mar. 2012, the entire contents of which areherein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to analytical chemistry, more particularlyto liquid chromatography, especially liquid chromatography with massspectrometry (LC-MS) and UV absorbance (LC-UV) detectors.

BACKGROUND OF THE INVENTION

Analytical methods based on high resolution liquid chromatographyseparation followed by mass spectrometry detection (LC-MS) or UVabsorbance (LC-UV) detection are widely used for the analysis of a widerange of compounds such as biotoxins, drugs, persistent environmentalpollutants, and other chemicals in wide range of samples such as plantand animal tissues, soil, water, etc. The identification of compoundspresent in samples is usually based on a match of both chromatographicretention time and mass or UV spectral data for authentic chemicalstandards with those of putative compounds observed in a sample.However, the absolute retention times of analytes can be highly variablebetween different laboratories and instruments, and even between days inthe same laboratory. This usually requires the analysis of chemicalreference standards on the same day each batch of samples is run toallow a good match of retention times for the conclusive identificationof potential contaminants. This approach increases the workload ofanalysts and the cost of analyses. In addition, not every laboratory canstock all standards in order to accomplish this task as analysts may beconcerned with the analysis of hundreds of possible analytes. It wouldbe helpful to have a better way of cataloging retention data so thatanalytes can be more easily identified through a match of retentiontimes without the use of in-house standards.

As an example, the above is particularly true with the analysis ofmarine and freshwater biotoxins. There are many different groups ofbiotoxins and within each group there can be many different structuralanalogues. Standards for many of these biotoxins are not commerciallyavailable and if they are, they can be very expensive. Analysts in thisfield face a difficult problem of determining which biotoxin analoguesmight be present in samples such as water, algae and shellfish.Similarly, for structural analogues or metabolites of pharmaceuticalsand environmental pollutants, very few laboratories have ready access tostandard compounds. This would be of concern to fields such as athletedoping control and monitoring of environmental samples and foodstuffs,among others.

Another issue of concern is related to the establishment of routineLC-MS analysis methods such as scheduled selected reaction monitoring,in which specific ion transitions are monitored over narrow windows thatencompass the analytes of interest. The first step in setting up such amethod is usually to perform an analysis of a mixture of all targetanalytes prior to establishing the windows. Again, the possible lack ofevery standard compound in a laboratory, as well as the extensive workrequired in this operation, presents problems to the analyst.

One way to correct for variations in retention data is to use “relativeretention times (RRT)”, in which analytes' retention times are measuredrelative to that of an internal standard compound. This method worksfairly well in isocratic LC (constant solvent composition) but not inthe more commonly-used gradient mode (changing solvent composition)because different LC instruments have different hold-up volumes in thegradient mixing system, which results in offsets in RRT values. The RRTvalues will also vary if there is any difference in the rate of changeof the gradient slope or in column dimensions.

A better way to report retention data is to use “retention index (RI)”values. In this procedure, a series of homologous reference compoundsare co-injected with the analytes. An interpolation of analyte retentiontimes into a fitted curve of the plot of retention time vs. retentionindex value for the reference compounds results in a retention indexvalue for each analyte.

The use of retention indices has been widely used in the field of gaschromatography. In this case, a series of n-alkanes is usually used asthe RI standards and the resulting interpolated indices are usuallyreferred to as “Kovats retention indices”. This is possible because thecommonly used flame ionization detector responds well to most organiccompounds, including the n-alkanes, albeit that all the compounds mustbe volatile. These are not applicable to LC, especially LC-UV or LC-MS,because the n-alkanes are not easily detected by common UV or MSdetectors.

Several different RI systems have been investigated by other researchersfor use in LC-UV analysis (see Scheme 1).

These are most commonly used with the UV absorbance detector. Theseinclude Smith's work on alkyl aryl ketones (Smith R. M., (1982) J.Chromatogr. 236, 313-320; Smith R. M., (1995) Journal of ChromatographyLibrary 57, 93-144), Baker and Ma's work on 2-ketoalkanes (Baker J. K.,Ma C-Y., (1979) J. Chromatogr. 169, 107-115) and Bogusz and Aderjan'swork on 1-nitroalkanes (Bogusz M., Aderjan R., (1988) J. Chromatogr.435, 43-53). Two journal papers by one group alluded to the use ofparabens (n-alkyl esters of 4-hydroxy benzoic acids) for measuringretention indices of phenols using LC-UV analysis (Yamauchi S., Mori,H., (1990) J. Chromatogr. A. 515, 305-311; Yamauchi S., (1993) J.Chromatogr. 635, 61-70). There have also been publications onapplication of LC-UV retention indices in the toxins field (Kuronen P.,(1989) Archives Environ. Contam. Toxicology 18, 336-48; Frisvad J.,Thrane U., (1987) J. Chromatogr. 404, 195-214; Hill D. W., Kelley T. R.,Langner K. J., Miller K. W., (1984) Analytical Chemistry 56, 2576-2579).There has been only one publication on the use of retention indices forLC-MS (Kostiainen R., Kuronen P., (1991) J. Chromatogr. 543, 39-47) andthis was based on 1-[p-(2,3-dihydroxpropoxy)phenyl]-1-alkanones(Scheme 1) as retention index standards. However, the Kostiainen methoduses a set of standards that are complicated to synthesize and notcommercially available, but more importantly are not well suited tomodern LC-MS methods based on electrospray (ESI) or atmospheric pressurechemical ionization (APCI).

Retention index standards of the prior art such as the alkylphenoneswere designed mainly for LC with UV absorbance detection. They are notwell suited to LC-MS methods based on electrospray (ESI) or atmosphericpressure chemical ionization (APCI), the most commonly used ionizationsystems. In-studies carried out by the inventors of the presentinvention, tests on alkylphenones as retention index standards in LC-MSshow that the sensitivity of the alkylphenones in positive ion ESI islow, which requires more concentrated solutions to be injected which inturn results in bad peak shapes in the chromatograms. Also, they cannotbe detected in the negative ion mode. Tests on parabens as retentionindex standards in LC-MS were more promising because they can bedetected in negative ion mode but overall, their performance was lackingin terms of sensitivity and they still require high concentrations to beinjected. In addition, there is potential susceptibility of the phenoliccompounds to retention time changes due to variations in pH of themobile phase, which can change the charge state of the parabens.

There remains a need for effective LC retention index standards,particularly for LC-MS methods.

SUMMARY OF THE INVENTION

It has now been found that a homologous series of neutrally chargedcompounds comprising at least one functional group bearing a positivecharge and at least one functional group bearing a negative charge areadvantageous retention index standards for liquid chromatography,especially for liquid chromatography-mass spectrometry (LC-MS) methods,more especially for LC-MS methods employing electrospray (ESI) oratmospheric pressure chemical ionization (APCI) ionization systems. Theterm “homologous series” is used to mean a group of compounds varying instructure only by the number of methylene groups in an alkyl chain.

Thus, there is provided a method of identifying an analyte of interestcomprising: introducing the analyte of interest together with a seriesof homologous retention index standards into a liquid chromatographysystem, the retention index standards comprising neutrally chargedcompounds comprising at least one functional group bearing a positivecharge and at least one functional group bearing a negative charge;assigning a retention index value to the analyte of interest based onretention times and retention index values of the retention indexstandards; and, comparing spectral data and the retention index value ofthe analyte of interest to a library of spectral data and the retentionindex values for known compounds to identify the analyte of interest.

There is further provided use of a neutrally charged compound comprisingat least one functional group bearing a positive charge and at least onefunctional group bearing a negative charge as a retention index standardin liquid chromatography.

There is further provided a series of retention index standards for usein liquid chromatography, the series of retention index standardscomprising two or more neutrally charged homologous compounds, eachcompound comprising at least one functional group bearing a positivecharge and at least one functional group bearing a negative charge. Theterm “series of retention standards” is used to mean a set of compoundsvarying in structure by the number of methylene groups in an alkylchain, which variation results in different retention times.

There is further provided a use of the series of retention indexstandards in accordance with the present invention for creating alibrary of retention indices for reference compounds.

There is further provided a kit comprising a series of homologouscompounds together with an indication or instructions for their use asretention index standards in liquid chromatography, each homologouscompound being a neutrally charged compound comprising at least onefunctional group bearing a positive charge and at least one functionalgroup bearing a negative charge.

The neutrally charged compounds useful as retention index standards inthe present invention have an overall neutral charge but bearindividually charged functional groups. One or more of the individuallycharged functional groups bears a permanent positive charge while one ormore bears a permanent negative charge provided the total charge of thecompound is zero. Separation of charge between ionized functional groupswithin the compound advantageously enhances ionization and sensitivityin mass spectrometry in both the positive and negative ion modes.Further, compounds having permanently ionized functional groups and anoverall neutral charge state will not vary with changes in mobile phasepH, thus making their retention times less sensitive to pH changes inthe mobile phase.

Furthermore, the retention index (RI) standards used in the presentinvention are more polar than those currently being used, which resultsin quicker elution of the most polar members of the RI standards on thereverse-phase columns typically used in liquid chromatography methods.Earlier elution of the standards permits bracketing of polar analytesthat are not bracketed by current non-polar standards, thereby extendingthe utility of the present retention index standards to a wider range ofanalytes.

The positively charged functional group preferably comprises aquaternary amine, tertiary amine, secondary amine, primary amine orimine group. Particularly preferred is a quaternary amine group. Thenegatively charged functional group preferably comprises a sulfonate,sulfate, phosphate, carboxyl, phenol, nitrate or arsenate group.Partcularly preferred is a sulfonate group. In a preferred embodiment,the compound comprises a quaternary amine group and a sulfonate group.In a particularly preferred embodiment, the compound comprises a singlequaternary amine group and a single sulfonate group.

In a preferred embodiment, the compounds are amine compounds of Formula(I):

where: m is an integer from 1 to 23; R₁ is H or CH₃; R₂ and R₃ areindependently C₁₋₂₃ straight or branched chain alkyl group, or R₂ and R₃taken together form a C₁₋₂₃ straight or branched chain alkyl imine groupwith the nitrogen atom, or R₂ and R₃ taken together form a 5- or6-membered aromatic or aliphatic heterocyclic ring with the nitrogenatom; p is an integer from 1 to 23; and X is H or a sulfonate, sulfate,phosphate, carboxyl, phenol, nitrate or arsenate group; and that thecompound comprises at least one of sulfonate, sulfate, phosphate,carboxyl, phenol, nitrate or arsenate group. Preferably, m is an integerfrom 1 to 17. Preferably, p is an integer from 0 to 17. Preferably, thelength of straight or branched chain alkyl or straight or branched chainalkyl imine group is C₁₋₁₇.

The 5- or 6-membered aromatic or aliphatic heterocyclic ring may besubstituted or unsubstituted. Where the ring possesses one or moresubstituents, the substituents are preferably independently sulfonate,sulfate, phosphate, carboxyl, phenol, nitrate, arsenate, C₁₋₂₃ straightor branched chain alkyl or C₁₋₂₃ straight or branched chain alkylsubstituted by sulfonate, sulfate, phosphate, carboxyl, phenol, nitrateor arsenate. The ring is preferably a pyridine or piperidine ring, morepreferably a pyridine ring. Compounds of Formula (I) are generally knownin the art or can be synthesized from readily available startingmaterials using known procedures for the formation of amine and iminecompounds.

In a particularly preferred embodiment, the compounds are1-alkylpyridinesulfonic acids (APSAs). Some particularly preferred APSAsare compounds of Formula (II):

where n is an integer from 0 to 23, preferably from 0 to 17. Thesulfonate group (SO₃) may be at the 2-, 3- or 4-position. Preferably,the sulfonate group (SO₃ ⁻) group is at the 3-position. A series ofhomologous compounds of formula II means a set of compounds of formulaII wherein only n is varied.

Compounds of Formula (II) may be synthesized by reacting apyridinesulfonic acid (e.g., 3-pyridinesulfonic acid (CAS RegistryNumber: 636-73-7)) with n-alkyl halides (CH₃(CH₂)_(n)L, where n is asdefined above and L is a leaving group, for example a halogen (e.g. Clor Br). Some examples of APSAs are also known in the art, for example,C10 (n=9) and C12 (n=11) homologues (Ainsworth C., et al (1967) J.Medicinal Chemistry 10(2), 158-161) and C8 (n=7) to C24 (n=23)homologues (U.S. Pat. No. 4,148,797; U.S. Pat. No. 4,501,673). TheN-methyl homologue (n=0) is commercially available. None of the knownAPSAs are known to be useful as retention index standards.

In the practice of the present invention, a series of homologouscompounds useful as retention index standards in accordance with thepresent invention are introduced into an LC system together with one ormore analytes of interest or a sample, for example by co-injection. Ifthe instrument provides sufficient retention time reproducibility, thenthe retention index standards may also be injected in one run and theanalytes of interest or a sample in a separate run. The retention indexstandards permit assignment of retention index (RI) values to the one ormore analytes. Thus, a retention index (RI) value for each analyte maybe obtained by interpolating analyte retention times into a fitted curveof the plot of retention time vs. retention index value for theretention index standards. The retention index value of the retentionindex standard is defined by the number of carbons in the longest alkylchain in the molecule. There should be at least two retention indexstandards in the series, but more than two is preferable. Preferably,the number of retention index standards in the series should be in arange of from about 4 to 16 homologues. The series of homologouscompounds used as the retention index standards preferably covers a widerange of masses to permit a good spread in retention times, therebyenabling effective bracketing of all analytes of interest andinterpolation of retention times for a wider range of analytes. The RIvalues of the analytes of interest in a sample may be used inconjunction with spectral data (for example, mass spectrometry or UVabsorbance data) on the same analytes to search a library (e.g.,database) or libraries of retention indices and/or spectra of referencecompounds for positive identification of known compounds in the sample.

The series of retention index standards may also be used to createlibraries (e.g. databases) of retention indices for reference compoundsfor use in liquid chromatography analyses. Such libraries can be usednot only for identifying analytes as discussed above, but also topredict retention times of the analytes on various columns using variousgradient conditions and on different LC instruments. Documentedretention indices for reference compounds in a library can facilitatethe establishment of retention windows of targeted analytes in order topermit the programming of a scheduled selected reaction monitoringmethod. The process would involve first performing an analysis of amixture of the retention index standards under the LC conditions andcolumns to be used for the sample. By interpolating the retentionindices of the reference compounds in the library using a plot ofretention time versus retention index for the retention index standards,the expected retention times of various reference compounds under therun conditions can be calculated.

The series of homologous compounds used as the retention index standardsmay be packaged into a kit together with an indication or instructionsfor their use as retention index standards in liquid chromatography. Thecompounds may be provided in the kit in separate containers and thenmixed in desired proportions to be introduced with the analyte into theLC system. Preferably, the compounds are pre-prepared and included inthe kit in ready-to-use mixtures of the retention index standards insolution.

The liquid chromatography (LC) system may be any suitable LC system.Liquid chromatography systems with mass spectrometry (LC-MS) or UVabsorbance (LC-UV) detectors are particularly preferred. The inventionis particularly useful for liquid chromatography-mass spectrometry(LC-MS) methods, more particularly for LC-MS methods employingelectrospray (ESI) or atmospheric pressure chemical ionization (APCI)ionization systems.

Analytes of interest may be, for example, toxins (e.g., biotoxins orother poisons), pharmaceuticals, drugs of abuse, peptides, persistentenvironmental pollutants, food contaminants, or any other compound ofinterest. Of particular interest are marine and freshwater toxins aswell as drugs used in aquaculture. Analytes may be from samples of anykind, for example, environmental samples (e.g., water, soil and air),tissue samples (e.g., plant or animal tissues), bodily fluids (e.g.,urine, blood, serum, semen), etc. Preparation of samples for use in LCmethods may be accomplished by generally known procedures in the art.

Further features of the invention will be described or will becomeapparent in the course of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, embodimentsthereof will now be described in detail by way of example, withreference to the accompanying drawings, in which:

FIG. 1 depicts an LC-MS analysis of a 1-alkyl-3-pyridinesulfonic acid(APSA) mixture containing C4 to C18 homologues (compounds of Formula (I)where n=3−17);

FIG. 2 depicts a spline fit graph for APSA standards with retentiontimes of APSAs plotted against retention index value and fitted with acubic spline curve;

FIG. 3 depicts ionization and fragmentation characteristics of APSAs;

FIG. 4 depicts LC-MS analysis of a cyanobacterial extract containingmicrocystins co-injected with a mixture of APSA standards, in which:upper plots show signals for targeted microcystins (not all the analytepeaks labeled) and bottom plot shows signals for the APSA standards (theAPSA peaks labeled with their retention index numbers, 400 to 1800);

FIG. 5 depicts a plot of retention time vs. retention index value forAPSA standards observed in the LC-MS analysis shown in FIG. 4 (circlesand solid trace) and for a separate analysis (triangles and dashedtrace) on the same LC column but with a 60 min gradient rather than the30 min used in FIG. 4;

FIG. 6 depicts LC-MS analysis of a mixture of reference standards ofvarious drugs that are routinely monitored by food inspection agenciesbecause of their use in agriculture and aquaculture;

FIG. 7 depicts a plot of the retention times vs. retention index valuesfor APSA standards observed in the LC-MS analysis shown in FIG. 6, witha cubic spline fit generated to fit the data; and,

FIG. 8 depicts a plot of the retention times vs. retention index valuesfor APSA standards measured in three different LC-MS analyses using twodifferent columns (150 vs. 50 mm length) and two different gradients,with cubic spline fits generated to fit the data.

DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to FIG. 1, an LC-MS analysis of a 1-alkyl-3-pyridinesulfonicacid (APSA) mixture containing C4 to C18 homologues demonstrates thatAPSAs give good chromatographic performance. With reference to FIG. 2,the plot of retention time vs. retention index for the APSA homologuesgives a curve that is well-fitted by a cubic spline. Retention index isdefined as the number of carbons in the side chain of the APSAmultiplied by 100. The APSA compounds give excellent response in massspectrometric detection using either positive or negative ion modes.Selected reaction monitoring (SRM) can be used to monitor the compoundsas they all fragment to the same product ion (m/z 160 in positive modeand m/z 158 in negative mode) (see FIG. 3). The compounds also have anexcellent UV chromophore with an absorbance maximum at 266 nm, so theycan be used as retention index markers in LC-UV analyses as well.

Example 1: Use of APSA Retention Index Standards to IdentifyMicrocystins by LC-MS

A sample of cyanobacterial extract containing analytes of interest (i.e.microcystins) was co-injected with a mixture of the C4-C18 APSAstandards into a reversed phase liquid chromatography-mass spectrometrysystem. The system comprises an LC-ESI-QqQ (QTRAP 4000) instrument withan Agilent 2.7 μm-Poroshell 120 Å SB-C18 column (2.1×150 mm). Elutionconditions comprise mobile phase: A=water, B=MeCN/water (95:5), bothwith 50 mM formic acid+2 mM ammonium hydroxide at pH 2.2; with agradient of 25-75% B over 30 min at a hold time of 5 min, flow rate of0.25 ml/min, and column temperature of 40° C. The liquid chromatography(LC) column provides separation of the complex mixture while the massspectrometer provides detection of both the microcystins within thesample and the co-injected APSA standards (FIG. 4). The characteristicmass spectral signals and the retention times of the microcystins andAPSAs in the sample are measured. In FIG. 4, the top box shows thesignals for targeted microcystins, while the bottom box shows signalsfor the APSA standards. The APSA peaks are labeled with their retentionindex numbers, 400 to 1800.

An interpolation of microcystin retention time into a fitted curve ofthe plot of retention time versus retention index value for the APSAs(FIG. 5) results in a measured retention index for the microcystins. InFIG. 5, the plot with circles and solid trace is for the LC-MS analysisshown in FIG. 4 (30 min gradient), while the plot with triangles anddashed trace is a separate analysis on the same LC column under the sameconditions but with a 60 min gradient rather than a 30 min gradient. Thecurve fitting is best done using the method of cubic splines, and FIG. 5shows the cubic spline fits generated to fit both sets of data.

The resulting retention index of the analyte of interest, as well as itsmass spectral data, can then be compared to a database previouslyestablished for a wide range of analytes in order to identify theanalyte of interest. In the present example, the sample contains morethan one analyte of interest (i.e., multiple microcystins) and themethod generates a list of analytes identified in the sample (Tables1a-1c).

TABLE 1a Short gradient (25-75% B, 30 min) Microcystin Precursor RTAverage 1 day SD 4 day SD Code Ion > 135 (min) Rla (n = 5) (n = 5, d =4) 3dm7dmRR 505.8 9.17 832.6 0.4 0.5 RR 519.8 9.64 847.2 0.3 0.8 7dmRR512.8 9.72 850.4 0.4 0.7 Nod-R 825.5 10.91 888.7 0.5 0.7 YR 1045.6 12.12929.3 0.4 0.6 7dmYR 1031.6 12.30 934.1 0.4 0.7 LR 995.6 12.69 946.7 0.11.3 7dmLR 981.6 12.87 953.3 0.3 1.3 3dm7dmLR 967.6 13.24 965.6 0.4 1.2(O—Me)LR 1009.9 13.61 977.8 0.5 1.3 7dmHiIR 995.6 13.82 985.2 0.8 0.9 RA953.8 14.16 995.3 0.6 2.1 LA 910.6 16.93 1092.5 0.2 2.4 isoLA 910.617.35 1106.8 0.3 2.9 LY 1002.9 17.91 1128.3 0.4 1.8 LW 1025.9 20.771232.9 0.2 1.8 LF 986.8 21.38 1255.1 0.3 2.4

TABLE 1b Long gradient (25-75% B, 60 min) Microcystin Precursor RTAverage 1 day SD 4 day SD Code Ion > 135 (min) Rlb (n = 5) (n = 5, d =4) 3dm7dmRR 505.8 11.33 842.3 0.7 1.2 RR 519.8 12.18 858.4 0.5 1.5 7dmRR512.8 12.32 862.0 0.3 1.2 Nod-R 825.5 14.29 901.3 0.2 1.1 YR 1045.616.75 947.0 0.4 0.8 7dmYR 1031.6 17.01 952.1 0.5 0.8 LR 995.6 17.75964.9 0.2 1.6 7dmLR 981.6 18.19 972.9 0.2 1.9 3dm7dmLR 967.6 18.85 984.70.5 1.3 (O—Me)LR 1009.9 19.59 997.6 0.4 1.5 7dmHiIR 995.6 19.94 1004.50.5 1.4 RA 953.8 20.45 1012.8 0.2 2.3 LA 910.6 25.63 1110.0 0.4 2.8isoLA 910.6 26.39 1124.4 0.2 3.0 LY 1002.9 27.68 1150.4 0.2 1.9 LW1025.9 33.20 1259.1 0.5 1.9 LF 986.8 34.25 1280.6 0.4 2.5

TABLE 1c 2-point correction of (b) to match (a) Microcystin CodePrecursor Ion > 135 Rlc % Diff. to (a) 3dm7dmRR 505.8 831.6 −0.11 RR519.8 847.2 0.00 7dmRR 512.8 850.8 0.04 Nod-R 825.5 888.9 0.03 YR 1045.6933.3 0.42 7dmYR 1031.6 938.3 0.44 LR 995.6 950.7 0.41 7dmLR 981.6 958.40.53 3dm7dmLR 967.6 969.9 0.43 (O—Me)LR 1009.9 982.4 0.46 7dmHiIR 995.6989.0 0.38 RA 953.8 997.1 0.18 LA 910.6 1091.0 −0.13 isoLA 910.6 1104.8−0.18 LY 1002.9 1129.9 0.14 LW 1025.9 1234.5 0.12 LF 986.8 1255.1 0.00

Table 1a provides retention times (RT) and retention indices (RI)measured for the microcystins detected in the LC-MS analysis shown inFIG. 4 (30 min gradient). Excellent reproducibility data for analyses isshown for within-day (number of runs, n=5) and between-day (number ofdays, d=4) runs. Also shown is data using the same column but with a 60min gradient (Table 1b). Finally, in Table 1c, the data from the 60 mingradient (Table 1b) has been adjusted to match the 30 min gradient data(Table 1a) using a 2-point correction based upon the retention data fortwo microcystins, RR and LF. This process allows fine tuning of thematching of data and results in less than 1% difference is observed forthe corrected 60 min RI data vs. the 30 min RI data (Table 1c).

Example 2: Use of APSA Retention Index Standards to Establish a Databaseof Retention Indices of Analytes for LC-MS

A solution containing various reference compounds (i.e. drugs that havebeen used in agriculture and aquaculture and that are routinelymonitored by food inspection agencies) is co-injected along with amixture of the C4-C18 APSA standards into a reversed phase liquidchromatography-mass spectrometry system. The system comprises anLC-ESI-QqQ (QTRAP 4000) instrument with an Agilent 2.7 μm-Poroshell 120Å SB-C18 column (2.1×150 mm). Elution conditions comprise mobile phase:A=water, B=MeCN/water (95:5), both with 50 mM formic acid+5 mM ammoniumhydroxide at pH 2.2; with a gradient of 5-100% B over 30 min at a holdtime of 5 min, flow rate of 0.25 ml/min, and column temperature of 35°C. The liquid chromatography (LC) column provides separation of thecomplex mixture while the mass spectrometer provides detection of boththe reference compounds and the co-injected APSA standards. Thecharacteristic retention times of the reference compounds and the APSAsare measured. FIG. 6 depicts the LC-MS analysis of the mixture ofreference compounds and co-injected APSA standards. The upper plots showthe signals for drugs (not all the drug peaks are labeled) while thebottom plot shows the signals for the APSA standards that wereco-injected with drugs.

An interpolation of the retention times of the reference compounds intoa fitted curve of the plot of retention time versus retention indexvalue for the APSAs (FIG. 7) results in a measured retention index forthe reference compounds. The curve fitting is done using the method ofcubic splines. The resulting retention index of the reference compoundsare then entered into a database, for example Table 2. Table 2 providesa listing of the reference compounds analyzed in FIG. 6 along with theirmeasured retention times (RT) and calculated retention indices (RI). Thestandard deviation (SD) value shows the excellent reproducibilitymeasured from 5 repeat analyses on the same day.

TABLE 2 Name of Compound Abbrev SRM transition Avg. RT (min) Avg. RI SD(n = 5) RI Sulfonamides Sulfanilamide SNA 173.1 > 92.1  2.70 379.7 1.3Sulfacetamide SAA 215.1 > 156.1 5.58 471.9 0.6 Sulfadiazine SDZ 251.1 >156.1 6.42 500.1 0.4 Sulfathiazole STZ 256.1 > 156.1 7.29 530.4 0.4Sulfapyridine SPR 250.1 > 156.1 7.34 531.9 0.2 Sulfamerazine SMR 265.1 >92.1  7.75 546.8 0.4 Sulfamethazzine SMZ 279.1 > 186.1 8.76 584.0 0.3Sulfamethizole SML 271.1 > 156.1 9.19 600.3 0.2 SulfamethoxypyridazineSMP 281.1 > 126.1 9.25 602.9 0.2 Sulfachloropyridazine SCP 285.1 > 156.110.52 653.9 0.3 Sulfadoxine SDO 311.1 > 156.1 11.07 676.9 0.3Sulfamethoxazole SMO 254.1 > 156.1 11.25 685.1 0.2 Sulfaquinoxaline SQO301.1 > 156.1 13.24 780.4 0.2 Nitroimidazoles 1-(2-Hydroxyethyl)-2-MNZ-OH 188.1 > 123.1 3.95 419.5 0.5 hydroxymethyl-5- nitroimidazole2-Hydroxymethyl-1- HMMNI 158.1 > 140.1 5.00 453.1 0.2methyl-5-nitrimidazole Metronidazole MNZ 172.1 > 128.1 5.13 457.3 0.5Dimetridazole DMZ 142.1 > 96.1  5.90 482.6 0.3 Ronidazole RNZ 201.1 >55.1  6.22 493.3 0.4 1-Methyl-2-(2′- IPZ-OH 186.1 > 168.1 8.93 590.6 0.2hydroxyisopropyl)-5- nitroimidazole Ipronidazole IPZ 170.1 > 109.1 11.07676.9 0.4 Fluoroquinolones Ciprofloxacin CIPRO 332.2 > 245.1 9.20 601.00.2 Danofloxacin DANO 358.2 > 96.1  9.54 614.1 0.3 Enrofloxacin ENRO360.2 > 316.2 9.82 625.2 0.4 Sarafloxacin SARA 386.2 > 342.2 10.82 666.20.3 Quinolones Oxolinic acid OXA 262.1 > 216.1 12.53 744.6 0.6Macrolides Erythromycin ERY 734.5 > 158.1 14.23 833.3 0.6 DyesLeucocrystal Violet LCV 374.4 > 358.1 10.68 660.3 0.6 Leucomalachitegreen LMG 331.1 > 316.1 16.76 983.2 0.9 Malachite Green MG 329.2 > 313.119.84 1180.8 1.2 Crystal Violet CV 372.4 > 356.1 23.31 1406.5 1.1Brilliant Green BG 385.3 > 341.1 25.90 1572.6 1.0

Example 3: Use of APSA Retention Index Standards to Predict RetentionTimes for Analytes in Order to Establish Windows for Scheduled SRMAnalysis by LC-MS

A database of retention indices for reference compounds (e.g., Table 2)is used to predict retention times of the same compounds that are to berun on columns with different dimensions, possibly with using variousgradient conditions or different LC instruments. This can facilitate theestablishment of retention windows of targeted analytes in order topermit the programming of a scheduled selected reaction monitoringmethod. The process involves first performing an analysis of a mixtureof the APSA retention index standards under the LC conditions and on thecolumn and instrument to be used for samples. Interpolation of thedatabase retention indices for reference compounds into a plot of theretention times vs. retention index values for APSA standards wouldallow the calculation of retention times expected under those newconditions.

FIG. 8 depicts a plot of the retention time vs. retention index valuesfor APSA standards measured in three different LC-MS analyses using twodifferent columns (150 vs 50 mm length) and two different gradients. Acubic spline fit has been generated to fit the data. Table 3 presentsthe prediction of drug retention times on a 50×2 mm column using theretention indices documented in in Table 2 and the plot shown in FIG. 8.

TABLE 3 SRM Database Predicted Drug Class Name of Compound Abbrevtransition RI RT (min) Sulfonamides Sulfanilamide SNA 173.1 > 92.1 379.7 0.8 Sulfacetamide SAA 215.1 > 156.1 471.9 2.3 Sulfadiazine SDZ251.1 > 156.1 500.1 2.9 Sulfathiazole STZ 256.1 > 156.1 530.4 3.7Sulfapyridine SPR 250.1 > 156.1 531.9 3.7 Sulfamerazine SMR 265.1 >92.1  546.8 4.1 Sulfamethazzine SMZ 279.1 > 186.1 584.0 5.2Sulfamethizole SML 271.1 > 156.1 600.3 5.6 Sulfamethoxypyridazine SMP281.1 > 126.1 602.9 5.7 Sulfachloropyridazine SCP 285.1 > 156.1 653.97.1 Sulfadoxine SDO 311.1 > 156.1 676.9 7.6 Sulfamethoxazole SMO 254.1 >156.1 685.1 7.8 Sulfaquinoxaline SQO 301.1 > 156.1 780.4 9.9Nitroimidazoles 1-(2-Hydroxyethyl)-2- MNZ-OH 188.1 > 123.1 419.5 1.4hydroxymethyl- 5-nitroimidazole 2-Hydroxymethyl-1- HMMNI 158.1 > 140.1453.1 2.0 methyl-5-nitrimidazole Metronidazole MNZ 172.1 > 128.1 457.32.0 Dimetridazole DMZ 142.1 > 96.1  482.6 2.5 Ronidazole RNZ 201.1 >55.1  493.3 2.8 1-Methyl-2-(2′- IPZ-OH 186.1 > 168.1 590.6 5.4hydroxyisopropyl)-5- nitroimidazole Ipronidazole IPZ 170.1 > 109.1 676.97.6 Fluoroquinolones Ciprofloxacin CIPRO 332.2 > 245.1 601.0 5.7Danofloxacin DANO 358.2 > 96.1  614.1 6.0 Enrofloxacin ENRO 360.2 >316.2 625.2 6.3 Sarafloxacin SARA 386.2 > 342.2 666.2 7.4 QuinolonesOxolinic acid OXA 262.1 > 216.1 744.6 9.2 Macrolides Erythromycin ERY734.5 > 158.1 833.3 10.9 Dyes Leucocrystal Violet LCV 374.4 > 358.1660.3 7.2 Leucomalachite green LMG 331.1 > 316.1 983.2 13.5 MalachiteGreen MG 329.2 > 313.1 1180.8 16.3 Crystal Violet CV 372.4 > 356.11406.5 19.3 Brilliant Green BG 385.3 > 341.1 1572.6 21.5

Other advantages that are inherent to the invention are obvious to oneskilled in the art. The embodiments are described herein illustrativelyand are not meant to limit the scope of the invention as claimed.Variations of the foregoing embodiments will be evident to a person ofordinary skill and are intended by the inventor to be encompassed by thefollowing claims.

The invention claimed is:
 1. A method of identifying an analyte ofinterest comprising: introducing the analyte of interest together with aseries of homologous retention index standards into a liquidchromatography system, the retention index standards comprisingneutrally charged compounds, each of the neutrally charged compoundscomprising at least one functional group bearing a permanent positivecharge and at least one functional group bearing a permanent negativecharge; assigning a retention index value to the analyte of interestbased on retention times and retention index values of the retentionindex standards; and, comparing spectral data and the retention indexvalue of the analyte of interest to a library of spectral data and theretention index values for known compounds to identify the analyte ofinterest.
 2. The method according to claim 1, wherein the at least onefunctional group bearing a positive charge comprises a quaternary aminegroup.
 3. The method according to claim 1, wherein the at least onefunctional group bearing a negative charge comprises a sulfonate group.4. The method according to claim 1, wherein the series of homologousretention index standards comprises from 4 to 16 homologues.
 5. Themethod according to claim 1, wherein the series of homologous compoundsis co-injected with the analyte of interest into the liquidchromatography system.
 6. The method according to claim 1, whereinassigning a retention index value to the analyte of interest comprisesinterpolating analyte retention times into a fitted curve of a plot ofretention time vs. retention index value for the series of homologousretention index standards.
 7. The method according to claim 1, whereinthe analyte of interest is a toxin, pharmaceutical, drug of abuse,peptide, persistent environmental pollutant, or food contaminant.
 8. Themethod according to claim 1, wherein the liquid chromatography systemcomprises a liquid chromatography-mass spectrometry system and thespectral data comprises mass spectrometry data.
 9. The method accordingto claim 1, wherein the liquid chromatography system comprises a liquidchromatography system with an ultraviolet absorbance detector and thespectral data comprises ultraviolet absorbance data.
 10. The methodaccording to claim 8, wherein assigning a retention index value to theanalyte of interest comprises interpolating analyte retention times intoa fitted curve of a plot of retention time vs. retention index value forthe series of homologous retention index standards.
 11. The methodaccording to claim 8, wherein the analyte of interest is a toxin,pharmaceutical, drug of abuse, peptide, persistent environmentalpollutant, or food contaminant.
 12. The method according to claim 8,wherein the at least one functional group bearing a positive chargecomprises a quaternary amine group.
 13. The method according to claim 8,wherein the at least one functional group bearing a negative chargecomprises a sulfonate group.